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| Product Name | Column-Based FFPE DNA Extraction Kit |
| Catalog No. | DREK-0016 |
| Description | A silica membrane based DNA extraction kit designed for the purification of genomic DNA from formalin-fixed, paraffin-embedded tissue sections. The kit uses a xylene-based deparaffinization step followed by proteinase K digestion and heat-induced reversal of formaldehyde crosslinks. The silica membrane binding and washing system effectively removes residual chemical fixatives, pigments, and tissue debris, yielding DNA suitable for PCR-based analysis and targeted sequencing. |
| Intended Use | Extraction of DNA from FFPE tissue sections for retrospective molecular analysis including mutation detection by Sanger sequencing, targeted next-generation sequencing, PCR-based genotyping, loss of heterozygosity analysis, and archival specimen characterization in cancer research and pathology studies. |
| Principle / Technology | FFPE tissue sections are treated with xylene to remove paraffin, followed by ethanol washes to remove xylene. The deparaffinized tissue is digested with proteinase K in an SDS-containing buffer at elevated temperatures (56°C followed by 90°C) to release DNA and reverse formaldehyde-induced protein-DNA crosslinks. After digestion, a binding buffer and ethanol are added, and DNA binds to the silica membrane. Wash steps remove contaminants, and purified DNA is eluted in a low-salt buffer. |
| Detection Method | UV spectrophotometry; Qubit fluorometric dsDNA quantitation; agarose gel electrophoresis; Bioanalyzer for fragment size distribution; qPCR with multi-size amplicons (100, 200, 400 bp) for amplifiability assessment; targeted NGS for functional quality evaluation |
| Sample Type | Formalin-fixed, paraffin-embedded tissue sections (5-20 µm thick); FFPE tissue curls; specimens fixed in 10% neutral-buffered formalin; compatible with a wide range of tissue types fixed for varying durations; performance may be reduced with prolonged fixation (>72 hours) or non-standard fixatives |
| Performance Range / Specifications | Input: 1-8 FFPE sections (10 µm thick); DNA yield: 50 ng to 5 µg (highly tissue-dependent); A260/A280: 1.6-1.9; DNA fragment size distribution: 100-500 bp peak; qPCR amplifiability (200 bp target): >50% success rate from sections >5 mm²; column binding capacity: 20 µg |
| Sensitivity / LOD | Recovers amplifiable DNA from a single 5 µm FFPE section; PCR detection of single-copy genomic targets from sections with >5 mm² tissue area; effective with >20-year-old archival blocks under optimized conditions |
| Specificity | Human genomic DNA with expected formalin-induced modification profile; no detectable bacterial DNA; minimal mitochondrial enrichment; successful STR profiling and gender identification by amelogenin PCR; no PCR inhibitors detectable after purification |
| Reaction Conditions / Protocol | Place FFPE sections in microcentrifuge tube; add 1 mL xylene; vortex vigorously; centrifuge at 14,000 rpm for 3 minutes; remove supernatant; wash pellet with 1 mL 100% ethanol; vortex; centrifuge; remove supernatant; repeat ethanol wash; air-dry pellet or vacuum dry briefly; add 180 µL tissue digestion buffer and 20 µL proteinase K; incubate at 56°C for 1 hour; transfer to 90°C for 1 hour for crosslink reversal; centrifuge briefly; add 200 µL binding buffer and 200 µL 100% ethanol; vortex; transfer to spin column; centrifuge at 8,000g for 1 minute; wash with 500 µL wash buffer AW1; centrifuge; wash with 500 µL wash buffer AW2; centrifuge 3 minutes; elute with 30-100 µL elution buffer; incubate 5 minutes; centrifuge; total time: 4-5 hours |
| Components / Formulation | Deparaffinization solvent (xylene alternative available upon request), 100% ethanol (user-supplied), tissue digestion buffer (Tris-HCl pH 8.0, EDTA, SDS), proteinase K solution (20 mg/mL), binding buffer (guanidine hydrochloride, Tris-HCl, sodium acetate), wash buffer AW1 concentrate, wash buffer AW2 concentrate, elution buffer (10 mM Tris-HCl pH 8.5, 0.1 mM EDTA), silica membrane spin columns with collection tubes |
| Storage Conditions | Digestion buffer and buffers at room temperature; proteinase K at 2-8°C; columns at room temperature; protect binding buffer from light; xylene stored in flammable solvent cabinet |
| Shelf Life | 18 months from date of manufacture; proteinase K 12 months at 2-8°C after opening; buffers stable 12 months after opening |
| Package Specifications | 50 preparations and 250 preparations; includes columns, proteinase K, and buffers; xylene and ethanol not supplied; xylene-free version available |
| Product Form | Silica membrane spin columns; liquid buffers; proteinase K solution; xylene or xylene-substitute for deparaffinization (depending on kit version) |
| Quality Control | Each lot validated with standardized FFPE tissue: DNA amplifiability at 100 bp and 200 bp targets confirmed; no PCR inhibition; A260/A280: 1.6-1.9; lot-to-lot yield consistency <20% CV on reference FFPE material; sequencing QC with >80% bases ≥Q30 |
| Key Features | Standardized FFPE DNA extraction protocol; effective deparaffinization system; dual-temperature incubation for optimal crosslink reversal; silica membrane efficiency for FFPE-derived DNA; suitable for archived specimens; consistent performance across common tissue types |
| Purity | A260/A280: 1.6-1.9 (variable due to formalin modification); A260/A230: >1.5; amplifiability confirmed by qPCR |
| Concentration | Elution volume 30-100 µL; DNA concentration 1-50 ng/µL (highly variable depending on tissue area and preservation); use fluorometric quantitation for accurate assessment |
| Activity / Unit Definition | Not applicable for FFPE DNA extraction kits |
| Molecular Weight | Not applicable; FFPE DNA is fragmented with a mode of 100-500 bp depending on fixation conditions and specimen age |
| Source / Origin | Silica membrane from quartz microfiber; proteinase K is recombinant; xylene is ACS grade; all aqueous buffers are molecular biology grade, nuclease-free |
| pH Range / Optimal pH | Digestion buffer pH 7.8-8.2; binding buffer pH 5.5-6.5; wash buffers pH 6.5-7.5; elution buffer pH 8.3-8.7 |
| Shipping Conditions | Ambient temperature for all components; xylene (if included) requires flammable liquid shipping classification; proteinase K shipped with cold packs |
| Expiration Date / Stability | 18 months shelf life; real-time stability at 0, 6, 12, 18 months; proteinase K activity >85% after 12 months; DNA recovery performance maintained through expiry; column capacity stable through shelf life |
| Regulatory / Compliance | For research use only; not for diagnostic use; suitable for retrospective research with appropriate IRB approval; xylene-containing kits require appropriate laboratory ventilation and chemical hygiene compliance |
| Compatibility | Purified DNA suitable for PCR, qPCR, Sanger sequencing, targeted NGS panels, and STR analysis; compatible with all major DNA polymerases; may require uracil-DNA glycosylase treatment for certain NGS applications to address cytosine deamination artifacts |
| Recommended Buffer System | Tris-EDTA-SDS digestion with proteinase K; guanidine hydrochloride binding buffer; ethanol-based wash system; Tris-EDTA elution |
| Application Notes / Precautions | Use appropriate PPE and work in fume hood when handling xylene; trim excess paraffin from tissue block before sectioning; use new microtome blade for each block to prevent cross-contamination; collect sections from areas with highest tumor cell content (if applicable); do not exceed 8 sections of 10 µm to avoid column overloading; if DNA appears degraded, reduce 90°C incubation to 30 minutes; elute in pre-warmed buffer for maximum yield; xylene-free version recommended for laboratories seeking to eliminate toxic solvent use |
| Batch-to-Batch Consistency | Between-lot yield CV <20% on FFPE reference material; qPCR Ct shift <1.5 cycles between lots for 200 bp target; column binding capacity ≥20 µg per lot; proteinase K activity specification 20-25 U/mg; buffer pH controlled to ±0.1 units |
For research use only, not for clinical use.
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